Increased number of autophagosomes in MFSD8/CLN7-depleted cells

cells and an increase in the unfolded protein response. It involves the engulfment of cytosolic proteins or whole organelles (e.g. ribosomes, mitochondria) by a double membrane (Klionsky and Codogno, 2013). These double-membraned vesicles, called autophagosomes, fuse with endosomes and finally with lysosomes to form autolysosomes where the autophagic cargo is released and subsequently degraded (Eskelinen and Saftig, 2009). Macroautophagy is the major cellular pathway for recycling damaged and aged mitochondria (Raben et al., 2009b) and is important for cell renewal, especially in non-dividing cells as neurons, where altered proteins and damaged organelles cannot be cleared by redistribution to daughter cells by cell division (Wong and Cuervo, 2010). Thus impairment of autophagy is implicated in the pathogenesis of neurodegenerative disorders (Komatsu et al., 2006; Nedelsky et al., 2008; Ventruti and Cuervo, 2007) and lysosomal storage disorders (Lieberman et al., 2012).

To analyze the role of CLN7 for macroautophagy, MFSD8/CLN7 mRNA was depleted by shRNA and the endogenous levels of the autophagic marker protein microtubule-associated protein1 light chain 3-II (LC3-II) were quantified by western blotting. In MFSD8/CLN7 shRNA transfected HEK293 cells a statistically significant 2.76-fold increase in LC3-II levels was detected compared to scr shRNA transfected control cells.

To confirm our results, we performed LC3-II immunoblotting with total homogenates from MFSD8/CLN7-depleted HeLa cells. In agreement with the results in HEK293 cells, increased amounts of LC3-II were observed in MFSD8/CLN7-depleted HeLa cells. The data indicate that increased numbers of autophagosomes are present in MFSD8/CLN7-depleted cultured cells. In agreement, increased numbers of GFP-LC3-II positive autophagosomes were detected in MFSD8/CLN7 down-regulated HeLa cells compared to scrambled shRNA transfected control cells in immunofluorescence experiments. Interestingly, increased numbers of GFP-LC3-II positive autophagosomes were also observed in MFSD8/CLN7 overexpressed HeLa cells.

Autophagy is induced under starvation conditions to provide metabolites for cellular survival (Cao et al., 2006). During a 2-hour and a 4-hour starvation period elevated numbers of GFP-LC3-II positive autophagosomes were detected in both

MFSD8/CLN7-depleted cells and in control cells. The data indicate that during nutrient withdrawal the formation of new autophagosomes occurs both in control and in MFSD8/CLN7-depleted cells. These results suggest that in the presence of amino acids, nutrients and growth factors only MFSD8/CLN7-depleted cells are unable to compensate for the loss of MFSD8/CLN7 leading to increased numbers of autophagosomes, whereas cells containing MFSD8/CLN7 show unchanged autophagosomes. Under starvation conditions even the control cells expressing CLN7 become unable to compensate the nutrient withdrawal and hence an increase is shown here, too. It was reported that lack of amino acids and growth factors induces autophagy generally (Ishibashi et al., 2009).

Increased levels of LC3-II could be caused by an impaired degradation in autolysosomes, decreased fusion of autophagosomes with lysosomes or an induction of macroautophagy (Klionsky et al., 2012). To discriminate between these possibilities expression levels of beclin were further analyzed. Beclin1 is a multifunctional protein and is known to have regulatory functions in autophagy (Wirawan et al., 2012). It is required for the formation of the PtdIns3K-beclin-1-complex and is essential for the induction of autophagy (Funderburk et al., 2010). Western blot analyses revealed that beclin1 levels were not significantly altered in MFSD8/CLN7-downregulated HEK293 cells. The data suggest that increased numbers of autophagosomes in MFSD8/CLN7-depleted cells are not due to an activation of autophagy, but rather a reduced removal/degradation of autophagosomes either by a block of fusion with lysosomes, altered mobility of autophagosomes or decreased degradation after fusion with lysosomes in autolysosomes. Increased LC3-II levels accompanied by unaltered beclin amounts were also observed in the brains of mouse models for mucolipidosis II (ML-II) (Kollmann et al., 2012), CLN2 disease (Micsenyi et al., 2013), CLN6 disease (Thelen et al., 2012) and CLN7 disease (L. Brandenstein, personal communication).

A gradual age-dependent increase in LC3-II and higher numbers of autophagosomes have also been reported in the brain of mouse models for CLN2 disease (Micsenyi et al., 2013), CLN3 disease (Cao et al., 2006), CLN6 disease (Thelen et al., 2012) and CLN10 disease (Koike et al., 2005; Shacka and Roth, 2007). However the changes in the number of autophagosomes, except for CLN10 disease, were mild and were mostly observed in older mice indicating a progressive accumulation of autophagosomes

during the course of the disease (Raben et al., 2009b). In addition, increased LC3-II levels have been observed for a number of lysosomal storage diseases like ML-II (Kollmann et al., 2012) and -IV (Vergarajauregui et al., 2008), Niemann Pick type C (Pacheco et al., 2007), Pompe disease (Raben et al., 2009a), multiple sulfatase deficiency, and mucopolysaccharidosis type IIIA (Settembre et al., 2008).

From this study it cannot be concluded whether the accumulation of autophagosomes in MFSD8/CLN7-depleted cells is due to decreased mobilization of autophagosomes, decreased fusion of autophagosomes with lysosomes or impaired lysosomal degradation (Wong and Cuervo, 2010). However, reduced lysosomal acidification, accumulation of storage material and decreased content or activity of lysosomal hydrolases have been reported to result in incomplete degradation of the content of autophagosomes. Based on the data of this study impaired acidification and lysosomal accumulation of storage material are unlikely to contribute to increased numbers of autophagosomes. However, alterations in lysosomal Ca ²⁺ levels have been shown to inhibit fusion of lysosomes with other organelles, mainly in the endocytic pathway (Luzio et al., 2007). Defects in the lysosomal Ca ²⁺ channel mucolipin 1 result in increased number of autophagosomes in neurons of mucolipin-1-deficient mouse brain (Curcio-Morelli et al., 2010) and in skin fibroblasts of MLIV patients (Vergarajauregui et al., 2008). It is possible that CLN7 may be involved in the export of divalent cations from the lysosomal lumen into the cytosol with Ca ²⁺, Zn ²⁺, Mn ²⁺ and Fe ²⁺ being potential substrates. The levels of MFSD8/CLN7 mRNA in the brain were reported to be altered by acute dietary iron loading and chronic iron accumulation in a mouse model in Hfe ^-/- mice, a mouse model for hemochromatosis (Johnstone et al., 2012; Johnstone and Milward, 2010). It is possible that CLN7 contributes to the transport of Fe ²⁺ across lysosomal membranes.

However, transport studies on isolated lysosomes from wild type and Cln7 knockout mice with Fe²⁺ as substrate have to be performed to show direct transport by CLN7.

In summary, the data obtained in MFSD8/CLN7-down-regulated HEK293 and HeLa cells and the CLN7 mouse model suggest that CLN7 disease has a macroautophagy inefficiency which is not due to a lysosomal acidification defect or the accumulation of storage material in lysosomes.

5.4 Comparison of MFSD8/CLN7-downregulated cells with Cln7-deficient fibroblasts from Mfsd8/Cln7-knockout mice

During the course of the studies an Mfsd8-lacZ gene trap and aMfsd8 knockout mouse model were generated by targeted disruption of the homologous Mfsd8/Cln7 gene by deletion of the critical exon 2 (S. Storch, personal communication, Damme et al., 2014).

The comparison of the data from this study and new data from the Mfsd8/Cln7-knockout mouse model indicate that the alterations of the protein composition of lysosomes was more pronounced in Cln7-deficient cells compared to MFSD8/CLN7-depleted cells. The data suggest that residual function of MFSD8/CLN7 caused by the incomplete depletion may weaken the phenotype in shRNA-mediated downregulated cells. In contrast to other CLN diseases (e. g. CLN1 disease, CLN2 disease), where different mutations in the same gene lead to a variety of phenotypes, defects in the MFSD8/CLN7 gene primarily result in the severe late-infantile NCL form suggesting that the majority of mutations lead to a complete loss of function. However, the cell culture model has some disadvantages compared to the mouse model. Increased numbers of autophagosomes in specific cell types (e. g. neurons in the brain) could be due to a long-term starvation period due to defective supply of monomeric compounds caused by lysosomal dysfunction with decreased degradation of macromolecules or export of catabolic monomers. It is unlikely, that a similar starvation occurs in cells cultured in full medium. Furthermore the rather late manifestation of CLN7 disease in patients suggest that either a critical threshold has to be exceeded or that other lysosomal membrane proteins with redundant functions can partially compensate for the loss of MFSD8/CLN7. In cerebellar precursor cells of cultured mouse Cln3Δex7/8 storage of subunit c could only be detected upon aging of cells at confluency (Fossale et al., 2004). The rather late manifestation of CLN7 disease in patients suggests that either a critical threshold has to be exceeded or that other lysosomal membrane proteins with redundant functions can partially compensate for the loss of CLN7.

5.5 Outlook

To date, only one hypomorphic mouse model for CLN7 disease exists which recapitulates partially the phenotype of human CLN7 patients (Damme et al., 2014). It will be of interest whether the complete knockout of the Cln7/Mfsd8 gene in mice leads to neurodegeneration in the brain and in the retina, accumulation of autofluorescent ceroid lipopigments, neuroinflammation and the activation of the lysosomal/autophagy machinery and on the biochemical level to lysosomal storage, missorting and altered processing of lysosomal enzymes, changed expression of lysosomal proteins and proteins involved in macroautophagy. The generated cell-based model of CLN7 disease is only partially valuable as a tool to study the cellular phenotype of CLN7 disease because a reduction of MFSD8/CLN7 by 75 % was achieved by shRNA-mediated transfection, leaving 25% of residual MFSD8/CLN7 mRNA. Phenotypic differences between the mild phenotype observed in hypomorphic Mfsd8/lacZ gene trap mice (Damme et al., 2014) and the more severe phenotype observed in Mfsd8 knockout mice (L. Brandenstein, personal communication) and the presence of late-infantile (majority of mutations) and juvenile forms (e. g. p.Ala157Pro) of CLN7 disease (Kousi et al., 2012) suggest that minor residual activity of wild type and mutant Cln7 might alter the onset and course of the disease and the cellular phenotype of CLN7-depleted cells. It is possible that the lack of cellular phenotypes in MFSD8/CLN7-depleted cells was due to residual amounts of wild type CLN7. Furthermore it is likely that both the cell type and the duration of CLN7 deficiency contribute to the pathological phenotypes observed in CLN7 disease.

In contrast to the neuropathological analyes of human CLN7 disease and the phenotype of the Mfsd8/lacZ mouse model, only consequences of short term depletion in mitotically active non-neuronal cells up to 96 hours were analyzed in this study. The data obtained in MFSD8/CLN7 down-regulated cells suggest that macroautophagy is impaired even in the presence of residual CLN7 levels, whereas the expression, biosynthetic sorting, and processing of cathepsin Z and D were not altered.

In the future interruption of the CLN7/MFSD8 gene by new techniques like the CRISPR/Cas9 genome editing system, has to be performed in HEK293 and HeLa cells to generate CLN7 knockout cells lines (Ran et al., 2013). With this technique a complete

knockout of MFSD8/CLN7 can be achieved in any cell line circumventing the problem of low residual CLN7 amounts. The analyses of these cells will clarify whether accumulation of autofluorescent ceroid lipopigments, increased levels of soluble lysosomal proteins, storage of subunit c of mitochondrial ATP-synthase in lysosomes and increased levels of autophagosomes can be observed in confluent CLN7-deficient cells upon aging. These knockout cell lines would contribute to study CLN7 disease-specific mechanisms.

6. Summary

1. Quantitative real-time PCR analyses showed low abundance of MFSD8/CLN7 mRNA in cultured HEK293 and HeLa cells. MFSD8/CLN7 mRNA expression was 8-fold higher in the human kidney derived cell line HEK293 compared to the HeLa cell line.

2. Comparison of siRNA- and shRNA-mediated down-regulation of MFSD8/CLN7 mRNA in HeLa and HEK293 revealed a higher reduction by shRNAs resulting in a decrease of MFSD8/CLN7 mRNA by 75% after 96 hours of expression compared to scrambled shRNA transfected cells.

3. Quantitative real-time PCR analyses of NCL-related and lysosomal genes in MFSD8/CLN7-depleted HeLa and HEK293 cells showed a reduction of CLN3, CLN6 and CLN8 mRNA levels. No significant changes in mRNAs coding for lysosomal proteins LAMP-1, lysosomal acid phosphatase, and cathepsin D in MFSD8/CLN7 down-regulated cells were detected suggesting no global transcriptional activation of lysosomal genes by TFEB in cells with short-term depletion of CLN7.

4. Endocytosis of the transferrin receptor at the cell surface and transport of transferrin-transferrin receptor complexes to endosomal compartments were unaltered in MFSD8/CLN7-downregulated HeLa cells.

5. In MFSD8/CLN7-depleted HEK293 cells decreased LAMP-1 and unaltered LAMP-2 protein levels were observed. In down-regulated HeLa cells amounts of LAMP-1 and LAMP-2 were unchanged and no gross changes in the number, size and distribution of LAMP-1 and LAMP-2 positive compartments were observed.

6. Biosynthetic sorting and processing of cathepsin D and cathepsin Z were unchanged in MFSD8/CLN7-depleted HeLa cells indicating that intracellular transport to lysosomes and lysosomal acidification were not impaired due to loss of CLN7.

7. Increased LC3-II amounts and an elevated number of autophagosomes in MFSD8/CLN7-depleted HEK293 and HeLa cells indicate an impairment of

macroautophagy. Unaltered beclin amounts showed that macroautophagy was not induced due to loss of CLN7.

8. In summary, the cell-based model for CLN7 disease generated by shRNA-mediated depletion did not replicate all changes observed in Cln7-deficient fibroblasts of a knockout mouse model for CLN7 disease. However, an increased number of autophagosomes was detected in CLN7-depleted cells making the cell-based model for CLN7 disease suitable to study macroautophagy. The data suggest that both the cell type and the duration of CLN7 deficiency contribute to the pathological phenotypes observed in CLN7 disease. In addition, residual amounts of wild type CLN7 protein may lead to the absence or later onset of cellular phenotypes.

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Im Dokument Generation and analysis of a cell-based model of CLN7 disease (Seite 86-106)